Bioresponsive Nanoparticles for the Intracellular Delivery of RNAi Therapeutics - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 140

nuclear engagement. The capability to alter the ratio of polymer
constituents allows determination of the correct HPR/NLS balance
required for successful nuclear pri-miRNA processing or cyto-
plasmic RISC-mediated siRNA silencing. Increased ratio of one
component may, however, compromise an essential property
provided by the other. These studies clearly highlight a structural-
activity relationship whereby polymer composition determines
intracellular compartmentalisation and consequent RNAi activity.

6.5 Hyperbranched System

This section describes the application of a bioreducible hyper-
branched polymer for nanoparticle-based delivery of RNAi triggers
[55]. This approach aims to modulate RNAi level and duration by
tuning the release rate of siRNA by altering the reducible disulphide
content within the polymer.
A series of rHB poly(amido amine) (PAMAM) polymers were
synthetized in the Oupicky laboratory [72] by reaction of trifunc-
tional amines to reducible bisacrylamide derivatives (Fig. 6.1B).
Michael addition copolymerization was performed using equal
molar ratio of triamine (1-(2-aminoethyl) piperazine) (AEPZ) and
bisacrylamide monomers, N,N 2-cystaminebisacrylamide (CBA), and

N,N 2-hexamethylenebisacrylamide (HMBA). Varying the ratio of the

reducible CBA to non-reducible HMBA allows production of a series
of polymers that contain different levels of disulphide bridges shown
to change the susceptibility to redox conditions in this polymer [73].
A prominent feature of the hyperbranched design is the high charge
density comprising of available free tertiary amines that facilitate
polyelectrolyte complex formation with anionic siRNA and secondary
and tertiary amines that allow endosomal escape by combined proton
sponge [74] and polymer-induced pore formation [75] mechanisms.
Extracellular nanoparticle stability is a prerequisite for siRNA
protection for nanoparticle transit in the bloodstream and cellular
uptake; however, intracellular disassembly is required to enable
RNAi engagement. These requirements can be fulfilled by exploiting
the redox potential gradient that exists between the extracellular
and intracellular environment as a consequence of the high levels
of reducing agents such as glutathione inside the cell compared to
plasma [71] that can be used to cleave redox-sensitive disulphide
bridges built into the bulk polymer. We investigated the relationship
between disulphide content, particle disassembly, siRNA release,
and RNAi activity.